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Fibrillin 5 Is Essential for Plastoquinone-9 Biosynthesis by
Binding to Solanesyl Diphosphate Synthases in Arabidopsis
Eun-Ha Kim,a Yongjik Lee,b and Hyun Uk Kima,1
a Department of Agricultural Biotechnology, National Academy of Agricultural Science, Rural Development Administration, Jeonju
54874, Republic of Korea
b Division of Integrative Biosciences and Biotechnology, Pohang University of Science and Technology, Pohang 37673, Republic of
Korea
ORCID ID: 0000-0002-4566-3057 (H.U.K.)
Fibrillins are lipid-associated proteins in plastids and are ubiquitous in plants. They accumulate in chromoplasts and
sequester carotenoids during the development of flowers and fruits. However, little is known about the functions of fibrillins in
leaf tissues. Here, we identified fibrillin 5 (FBN5), which is essential for plastoquinone-9 (PQ-9) biosynthesis in Arabidopsis
thaliana. Homozygous fbn5-1 mutations were seedling-lethal, and XVE:FBN5-B transgenic plants expressing low levels of
FBN5-B had a slower growth rate and were smaller than wild-type plants. In chloroplasts, FBN5-B specifically interacted with
solanesyl diphosphate synthases (SPSs) 1 and 2, which biosynthesize the solanesyl moiety of PQ-9. Plants containing
defective FBN5-B accumulated less PQ-9 and its cyclized product, plastochromanol-8, but the levels of tocopherols were not
affected. The reduced PQ-9 content of XVE:FBN5-B transgenic plants was consistent with their lower photosynthetic
performance and higher levels of hydrogen peroxide under cold stress. These results indicate that FBN5-B is required for PQ-9
biosynthesis through its interaction with SPS. Our study adds FBN5 as a structural component involved in the biosynthesis of
PQ-9. FBN5 binding to the hydrophobic solanesyl moiety, which is generated by SPS1 and SPS2, in FBN5-B/SPS homodimeric
complexes stimulates the enzyme activity of SPS1 and SPS2.
INTRODUCTION
Fibrillins are lipid-associated proteins found in all photosynthetic
organisms, from cyanobacteria to plants (Pozueta-Romero et al.,
1997; Kessler et al., 1999; Ytterberg et al., 2006; Simkin et al.,
2007; Cunningham et al., 2010). They were first identified in fibrils,
the thread-like structures involved in carotenoid storage in the
chromoplasts of bell pepper (Capsicum annuum) fruit (Newman
et al., 1989; Deruère et al., 1994), and were called fibrillins because
of their high abundance in fibrils. Members of the fibrillin protein
family have been given many different names over the years,
including plastid-lipid-associated protein (Pozueta-Romero
et al., 1997; Ting et al., 1998), chromoplast-specific carotenoidassociated protein (Vishnevetsky et al., 1996), and plastoglobulins
(Kessler et al., 1999). Recently, Singh and McNellis (2011) proposed that this type of proteins be called fibrillins, abbreviated FBN.
Fibrillins can be divided into 12 subfamilies, ranging from algae
to plants (Singh and McNellis, 2011). The fibrillin proteins appear
quite diverse, with molecular masses in the range of 21 to 42 kD,
pI values of 4 to 9, hydrophobic profiles, and plastid localization.
Fibrillins are distributed throughout the stroma or are associated
with the thylakoid membranes and with lipid globules (Vidi et al.,
2006; Lundquist et al., 2012). This diversity suggests that each
1 Address
correspondence to [email protected].
The author responsible for the distribution of materials integral to the
findings presented in this article in accordance with the policy described
in the Instructions for Authors (www.plantcell.org) is: Hyun Uk Kim
([email protected]).
www.plantcell.org/cgi/doi/10.1105/tpc.15.00707
fibrillin family member has specific biological functions (Laizet
et al., 2004; Singh and McNellis, 2011; Lundquist et al., 2012).
Arabidopsis thaliana contains 14 genes with sequence similarity to
fibrillin genes. All the fibrillins of Arabidopsis share three blocks of
similarity in the mature proteins (Laizet et al., 2004) and contain
hydrophobic domains that associate with lipids. Singh and
McNellis (2011) showed that the 14 fibrillins expressed in Arabidopsis contain a predicted lipocalin motif 1 within conserved
block 1 and suggested that the fibrillin family is involved in binding
to and transporting a range of small hydrophobic molecules. The
residues near the fibrillin C terminus, including aspartic acid, are well
conserved in the fibrillins (Singh and McNellis, 2011).
Fibrillins play a structural role in the formation of lipoprotein
structures in plastids. Deruère et al. (1994) demonstrated that
a 32-kD protein, designated fibrillin, purified from the chromoplasts
of a ripening bell pepper fruit, reconstituted the fibril structure in vitro
when it was combined with carotenoids and polar lipids. Furthermore, the overexpression of a pepper fibrillin in tobacco
(Nicotiana tabacum) increased the numbers of plastoglobules
(Rey et al., 2000). In addition to their structural roles, fibrillins are
involved in photosynthesis during plant development, tolerance
of photooxidative stress, and resistance to biotic stress (Rey
et al., 2000; Leitner-Dagan et al., 2006; Simkin et al., 2007;
Cunningham et al., 2010; Singh et al., 2010; Singh and McNellis,
2011). Recently, fibrillin mutants of Arabidopsis have extended our
understanding of the functions of the fibrillin proteins. The Arabidopsis FIBRILLIN1 (FBN1) gene family is involved in abscisic
acid-mediated protection from photoinhibition (Yang et al., 2006),
and the FBN1 and FBN2 gene families modulate jasmonate
biosynthesis during light and cold stress (Youssef et al., 2010).
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Apple tree (Malus domestica) and Arabidopsis lacking FBN4 are
susceptible to abiotic and biotic stresses, which are accompanied
by lower levels of plastoquinone-9 (2,3-dimethyl-6-solanesyl-1,4benzoquinone; PQ-9) in their plastoglobules. It has been suggested that FBN4, with its lipocalin domain, is involved in the
transport of PQ-9 and other molecules from the thylakoids to the
plastoglobules (Singh et al., 2010, 2012). However, the precise
biological functions of the fibrillins require further investigation.
In plants, the lipid-soluble plastid-localized electron carrier PQ-9
is essential for oxygenic photoautotrophs. It is involved in
photosynthetic electron transport and acts as a mobile redox
carrier (Trebst, 1978) and a cofactor in the desaturation of phytoene.
Consequently, PQ-9 functions in carotenoid biosynthesis
(Norris et al., 1995). Reduced PQ-9 also has antioxidant activity
in plants under stress conditions (Kruk and Trebst, 2008;
Szymańska and Kruk, 2010; Nowicka and Kruk, 2012). Furthermore, the redox state of PQ-9 mediates a number of photosynthetic
responses, such as state transitions, photosystem stoichiometry,
and the biosynthesis of the proteins of the photosynthetic apparatus
(Allen, 1995; Maxwell et al., 1995; Melis et al., 1996; Pfannschmidt
et al., 2001). In chloroplasts, PQ-9 is located in the thylakoids,
where it occurs in the free form or is associated with the QA and QB
sites of photosystem II (PSII). These forms of plastoquinone (PQ)
are likely part of the photoactive PQ pool and participate in both
electron transport and antioxidant functions (Kruk and Karpinski,
2006). PQ-9 is also found in plastoglobules and the inner membrane of plastids (Tevini and Steinmüller, 1985; Eugeni Piller et al.,
2012; Singh et al., 2012). These PQ-9 molecules are thought to be
nonphotoactive, storage forms that are used to replenish the
photoactive PQ pool (Zbierzak et al., 2010; Singh et al., 2012).
Previous studies have shown that PQ-9 biosynthesis occurs in
the membranes of the plastid envelope (Hutson and Threlfall,
1980; Soll et al., 1980, 1985; Joyard et al., 2009).
The biosynthetic pathways and enzymes involved in the production of PQ-9 and tocopherols in plants have been clarified
with genetic, genomic, and biochemical studies in Arabidopsis
(DellaPenna and Pogson, 2006; Sadre et al., 2006; Block et al.,
2013). Homogentisate (HGA) is the common head group of PQ-9
and tocopherols in plants. PQ-9 biosynthesis shares the formation
of the aromatic HGA ring with the tocopherol pathway. Whereas
HGA is condensed with phytyl diphosphate by homogentisate
phytyltransferase (VTE2) in tocopherol biosynthesis, the first
committed step in PQ-9 biosynthesis is the condensation of
homogentisate and solanesyl diphosphate (SPP), which is catalyzed by homogentisate solanesyltransferase (HST). HST uses HGA
(derived from the shikimate pathway) and SPP (biosynthesized in
the plastid nonmevolonate isoprenoid pathway) as substrates and
catalyzes their decarboxylation and prenylation to yield 2-methyl6-prenyl-1,4-benzoquinol (MSBQ). MSBQ is then methylated by
MSBQ/2-methyl-6-phytyl-1,4-benzoquinone methyltransferase
(VTE3) to yield PQ-9, which is cyclized into plastochromanol-8
(PC-8) by tocopherol cyclase (VTE1). PC-8 and the tocopherols
are lipid-soluble antioxidants essential for seed desiccation and
quiescence in Arabidopsis (Mène-Saffrané et al., 2010). The
solanesyl moiety is generated by the trans-type consecutive
condensation of isopentenyl diphosphate (IPP; C5) with farnesyl diphosphate (C15) or geranylgeranyl diphosphate (GGPP;
C20) and is catalyzed by solanesyl diphosphate synthases
(SPSs) (Hirooka et al., 2003). The SPSs of Arabidopsis (SPS1
and SPS2), tomato (Solanum lycopersicum) (SlSPS), and rice
(Oryza sativa) (OsSPS2) produce SPP in vitro, and the enzymes
are targeted to the plastids in planta (Jun et al., 2004; Ohara et al.,
2010; Block et al., 2013; Jones et al., 2013). The corresponding
single or double knockout mutants of Arabidopsis display either
reduced or absent production of PQ-9 and PC-8 (Block et al.,
2013). All three SPS enzymes of Arabidopsis function as homodimers (Hirooka et al., 2003; Jun et al., 2004; Hsieh et al.,
2011).
In this study, we identified and characterized an fbn5-1 T-DNA
insertion mutant of Arabidopsis with a seedling-lethal phenotype.
FBN5 is a phylogenetically distant relative of other fibrillins in
Arabidopsis (Singh and McNellis, 2011), and it localizes to the
stroma, with a lower pI and lower hydrophobicity than the other
fibrillins (Lundquist et al., 2012). Therefore, FBN5 may play different roles from those previously reported for the fibrillins that
localize primarily to the plastoglobules (Trebst, 1978; Yang et al.,
2006; Singh et al., 2010, 2012; Youssef et al., 2010). Surprisingly,
we found that FBN5 specifically interacts with two solanesyl diphosphate synthases, SPS1 and SPS2, in the chloroplasts. A
deficiency of FBN5 results in reduced levels of PQ-9 and PC-8 in
the leaves, suggesting that FBN5 plays a critical role in PQ-9
biosynthesis and is therefore essential for plant development and
growth. We propose a model for PQ-9 biosynthesis in plants in
which FBN5 functions in concert with SPS.
RESULTS
Mutation of FBN5 Results in Seedling-Lethal Phenotype
In this study, we investigated the function of the FBN proteins by
identifying homozygous mutants among T-DNA insertion lines
from the TAIR database (http://www.arabidopsis.org). Among 11
T-DNA insertion mutants of FBN genes, seedlings homozygous
for mutations in FBN5 were seedling-lethal, which was an unexpected phenotype for a mutated structural protein. Plants of
the T-DNA insertion line SALK_064597, which has an insertion in
exon 1 of FBN5 (Figure 1A), were grown in soil and genotyped by
PCR analysis using primers specific for the T-DNA and FBN5
(Figure 1B). Of 12 plants tested, three were wild type and nine were
heterozygous for the T-DNA insertion. No homozygous mutant
plants were identified. These results suggest that the homozygous
mutant is embryo- or seedling-lethal. When progeny seeds from
one of the heterozygous plants, designated FBN5/fbn5-1, were
germinated on agar medium in the absence of sucrose, approximately one-quarter of the seedlings quickly turned white and
stopped growing (Figure 1C). Murashige and Skoog (MS) medium
containing 1% sucrose allowed some growth of the seedlings,
but they eventually died.
To test whether the seedling-lethal phenotype cosegregated
with the fbn5-1 mutation, 120 progeny plants obtained from one
heterozygote on medium containing 1% sucrose segregated
in a green:white phenotypic ratio of 97:23. Among these plants,
10 randomly selected white seedlings were homozygous for
the fbn5-1 mutation and 12 randomly selected green seedlings
were either wild type or heterozygous at the FBN5 locus. Taken
FBN5 Is Essential for Synthesis of PQ-9
Figure 1. Identification and Characterization of an fbn5-1 Homozygous
Plant.
(A) Structure of the FBN5 locus showing the location of the T-DNA insertion in the fbn5-1 line (SALK_064597). Exons are shown with boxes;
introns are shown with lines. The locations of the oligonucleotide primers
used to genotype the fbn5-1 mutant and for RT-PCR are indicated.
(B) Genotyping of wild-type and fbn5-1 T-DNA insertion mutant plants by
PCR. A 1.2-kb PCR product was detected using the LP + RP primers, but
not with the LBa1 + RP primers in wild-type segregants. In fbn5-1 segregants, a 1-kb band was detected using the Lba1 + RP but not the LP + RP
primers.
(C) The fbn5-1 mutation is seedling-lethal. fbn5-1 seedlings germinated on
agar medium lacking sucrose, but stopped growing and became chlorotic.
Limited growth of fbn5-1 plants on medium containing 1% sucrose.
Bars = 1 cm.
(D) RT-PCR indicates that the fbn5-1 plant is a null mutant. Full-length
FBN5-A and FBN5-B transcripts, amplified with the P1 + P3 primers and
P1 + P2 primers, respectively, were detected in wild-type segregants but
not in fbn5-1 plants. Amplification of the ACTIN gene (ACT7, At5g09810)
was a control. Molecular mass markers are shown in the left lane of each
panel.
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together, these results indicate that the seedling-lethal phenotype
segregates as a single, recessive mutation (23 of 120; x2 = 2.17; P >
0.95) and cosegregates with the fbn5-1 T-DNA insertion.
The FBN5 locus expresses two differentially spliced transcripts
according to the TAIR database. We designated the shorter
variant FBN5-A and the longer variant FBN5-B (Supplemental
Figure 1A). FBN5-A differs from FBN5-B in the deletion of 14 amino
acids at the COOH terminus and the substitution of 10 amino acids
in the remaining part of the COOH terminus by alternative splicing
(Supplemental Figures 1B and 1C). The fibrillin domain occurs at
residues 85 to 267 of the FBN5-B polypeptide (Supplemental
Figures 1B and 1C). PROSITE (http://expasy.org) did not identify
a lipocalin motif in FBN5, whereas FBN4 was predicted to contain
the first motif of lipocalin, which is the most highly conserved in the
lipocalin proteins (Singh and McNellis, 2011). FBN5 contains
a region similar (53.8% identity; 84.6 similarity) to the lipocalin
motif 1 in FBN4 (Supplemental Figure 1C). This possible lipocalin
motif 1 in FBN5 has an invariant glycine, followed by an invariant
tryptophan and an aromatic residue (tyrosine), which are the
characteristically conserved residues of lipocalin motif 1 (Flower
et al., 1993).
RT-PCR analysis revealed that the FBN5-A and FBN5-B
transcripts were undetectable in the RNA from fbn5-1 plants,
whereas they were detected in the wild type (Figure 1D). The two
transcripts showed similar expression patterns in most tissues,
and the transcript levels were highest in the photosynthetic tissues, including in seedlings, developing leaves, stems, and unopened flowers (Supplemental Figure 2A). To further investigate
the expression of FBN5, we cloned a 2.7-kb fragment upstream
from the FBN5 initiation codon, fused it to the GUS reporter gene,
and used the construct to transform wild-type Arabidopsis plants.
GUS activity was strong in the young seedlings, developing roots,
developing leaves, and young stems and sepals of the flowers, but
was not detectable in siliques or petals (Supplemental Figure 2B).
To confirm that the fbn5-1 null mutation is directly responsible
for the seedling-lethal phenotype, FBN5-A and FBN5-B cDNAs
under the control of the CaMV 35S promoter were constructed and
used to transform heterozygous FBN5/fbn5-1 plants. Thirty-two
BASTA-resistant transformants showing a healthy, green phenotype were selected from each group (FBN5-A and FBN5-B).
Among the FBN5-A transgenic plants, 11 and 21 plants were
genotyped as wild type and fbn5-1/FBN5, respectively; no fbn5-1
plants contained the FBN5-A cDNA. Among the 32 FBN5-B
healthy green transformants, 18 were wild-type plants, eight were
fbn5/FBN5 plants, and six were genotyped as fbn5-1 plants. The
FBN5-B-complemented homozygous fbn5-1 plants showed
healthy green growth, similar to that of wild-type plants in soil
(Supplemental Figures 3A and 3B). This indicates that the FBN5-B
cDNA complemented the seedling-lethal phenotype but the
FBN5-A cDNA did not. Genomic DNA PCR using the P1 and P2
primers generated products of ;1.7 and 0.8 kb from the wild-type
and fbn5-1+35S:FBN5-B plants, respectively, indicating that
the latter were true fbn5-1 plants transformed with FBN5-B cDNA.
The expression of FBN5-B was higher in both the cauline and
rosette leaves of the fbn5-1+35S:FBN5-B plants than in those of the
wild-type plants (Supplemental Figures 3C and 3D). Collectively,
these results indicate that the fbn5-1 mutation causes the seedlinglethal phenotype and that the FBN5-B gene product is required for
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autotrophic growth. The maximum photosynthetic performance
of PSII was lower in the leaves of the fbn5-1 plants (Fv/Fm = 0.65)
than in those of the wild-type plants (Fv/Fm = 0.8) grown on
medium containing 1% sucrose. The fbn5-1 plants also accumulated more H2O2 than the wild-type plants (Supplemental Figure 4).
FBN5 Localizes in the Chloroplasts
It was previously reported that FBN5 is present in the stroma of
chloroplasts (Lundquist et al., 2012). To confirm that FBN5 is
targeted to the chloroplasts, we constructed GFP fusions with
FBN5-A and FBN5-B cDNA under the control of the CaMV 35S
promoter for transient expression in protoplasts derived from
Arabidopsis leaves. The GFP signal in the transformed protoplasts colocalized with the autofluorescence of the chlorophylls
in the chloroplasts (Figure 2A). The soluble and pellet fractions
were collected from the transformed protoplasts, and the FBN5
fusion proteins were detected with an anti-GFP antibody. Both
the FBN5-B:GFP and FBN5-A:GFP isoforms were detected, with
approximately equal amounts of preprotein and mature protein in
the pellet fractions of the protoplasts. It seems that the transient
overexpression of FBN5 produced high levels of preproteins,
which were probably improperly targeted to the plastid. Interestingly,
FBN5-B:GFP was the only mature form detected in the soluble
fraction (Figure 2B). These results indicate that the functional FBN5-B
protein is targeted to the chloroplast and processed into the mature
protein in the soluble portion of the organelle.
FBN5 Mutation Reduces the Levels of PQ-9 and PQ-8
Figure 2. Localization of FBN5 in Chloroplasts.
We used HPLC to determine the amounts of PQ-9, tocochromanols, carotenoids, and chlorophylls in the mutant and wild-type
plants because the photosynthetic capacity of the fbn5-1 plants
was reduced and FBN4 is involved in plastoquinone accumulation in the plastoglobules (Singh et al., 2012). Although fbn5-1 is
seedling-lethal when plants are grown in soil, seedlings cultivated on agar medium supplemented with 1% sucrose were pale
green, survived for several weeks, and produced sufficient tissue
for biochemical analyses. The amount of PQ-9 was reduced
;28-fold in the leaves of the fbn5-1 plants compared with that in
the wild-type plants of a similar age (Figure 3A). We did not detect
any PC-8, which is the product of PC-9 cyclization, in the leaves
of the fbn5-1 plants, unlike in the wild-type plants (Figure 3A). The
tocopherol (TC) profile in the leaves of the wild-type plants consisted of a-TC, g-TC, and d-TC in an ;94:3:3 ratio. In the fbn5-1
leaves, the levels of d-TC and g-TC were elevated relative to those in
the wild-type plants, whereas a-TC and the total tocopherols were
not significantly different (Figure 3B). Thus, it seems that the incompletely developed fbn5-1 plants accumulated higher levels of
d-TC and g-TC. The levels of lutein and b-carotene in the fbn5-1
plants were reduced by ;14 and 46%, respectively, compared with
those in the wild-type plants, whereas neoxanthin and violaxanthin
were only slightly reduced (Supplemental Figure 5A). The level of
antheraxanthin was increased in the fbn5-1 plants, indicating that they
were more photostressed than the wild-type plants (Supplemental
Figure 5A). Both chlorophylls were significantly reduced in the
fbn5-1 plants compared with the wild-type plants (Supplemental
Figure 5B).
(A) Subcellular localization of the FBN5-A:GFP and FBN5-B:GFP fusion
proteins. A sequence encoding GFP was fused to the 39 ends of the fulllength FBN5-A and FBN5-B cDNAs, which were transcribed from the
CaMV 35S promoter. Transient expression of these constructs in protoplasts derived from Arabidopsis mesophyll cells resulted in colocalization
of GFP fluorescence patterns with the autofluorescence of chlorophyll.
Bars = 20 µm.
(B) SDS-PAGE of proteins in the soluble (S) and pellet (P) fractions of
protoplasts expressing FBN5-B:GFP or FBN5-A:GFP fusion proteins and
immunoblots showing the localization of FBN5 proteins in the soluble and
pellet fractions of protoplasts.
To investigate whether the lack of PQ-9 and PC-8 is FBN5
mutation specific or is just one of the pleiotropic defects occurring
in any pale-green mutant, we assessed the levels of PQ-9, tocochromanols, carotenoids, and chlorophylls in four known palegreen mutants. The tested mutants were defective in nuclear
genes encoding essential chloroplast proteins, namely, PDS2
(encoding homogentisate solanesyltransferase), GDC1 (encoding
grana-deficient chloroplast 1), SBP (encoding the Calvin-Benson
cycle enzyme sedoheptulose-1,7-bisphosphatase), and AAE14
(encoding acyl-activating enzyme 14) (Tian et al., 2007; Kim et al.,
2008; Cui et al., 2011; Liu et al., 2012; Savage et al., 2013). The
gdc1-2, sbp-1, and aae14-1 pale-green homozygous mutants
were null, whereas the pds2-1 homozygous mutant was leaky
because the T-DNA insertion disrupted its promoter region
(Supplemental Figure 6). Consistent with previous findings, all of
FBN5 Is Essential for Synthesis of PQ-9
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Figure 3. Quantification of PQ-9 and Tocochromanols in Arabidopsis Leaves.
Reverse-phase HPLC was used to analyze total lipids in the leaves of 3-week-old wild-type (WT) and fbn5-1 plants grown on MS medium (+1% sucrose) ([A]
and [B]) and in 6-week-old wild-type and transgenic plants grown in soil ([C] and [D]). The quantities of individual tocopherols, PQ-9, and PC-8 were
determined relative to standards. Asterisks represent significance relative to the wild type by Student’s t test: **P < 0.01 and ***P < 0.001. Data are means 6 SD
(n = 3 to 4). N.D., not detected; FW, fresh weight.
these pale-green mutants exhibited pale-green cotyledons and
true leaves, with slow growth on medium containing sucrose
(Supplemental Figure 6). The total tocopherols in these pale-green
mutants did not differ from those in the wild-type plants, although
the compositions of their tocopherols varied (Supplemental
Table 1). However, all of the pale-green mutants showed significantly reduced levels of PQ-9 and PC-8: in order of severity,
pds2-1 > sbp-1 > aae14-1 > gdc1-2 plants. The total carotenoids
and chlorophylls were also significantly reduced in the plants
(aae14-1 > gdc1-2 > sbp-1 > pds2-1 plants) (Supplemental Table 2).
Therefore, it is likely that lower levels of PQ-9, PC-8, carotenoids,
and chlorophylls in the pale-green mutants are pleiotropic defects,
possibly attributable to the limited accumulation of the photosynthetic machinery. However, the extent of the reductions in
these molecules seems to be related to the specific mutation.
Moreover, the reductions in PQ-9 and PC-8 did not correlate with
the reductions in the carotenoids and chlorophylls in the palegreen mutants. For example, gdc1-2 plants seemed to be more
affected in the accumulation of carotenoids and chlorophylls than
in the accumulation of PQ-9, whereas the converse was true of
pds2-1 plants. The aae14-1 plants showed the lowest levels of
carotenoids and chlorophylls, but accumulated more PQ-9 and
PC-8 than the sbp-1, pds2-1, or fbn5-1 plants. In particular, the
pds2-1 plants, which are directly defective in PQ-9 biosynthesis
(Norris et al., 1995; Tian et al., 2007), showed similar levels of PQ-9
and PC-8, as well as total carotenoids and chlorophylls, to those of
the fbn5-1 plants. These results suggest that the lack of PQ-9 and
PC-8 in the fbn5-1 plants is specific for the FBN5 mutation.
Because the fbn5-1 mutation was seedling-lethal, we studied
the function of FBN5 by generating transgenic fbn5-1 plants with
an FBN5-leaky phenotype. A b-estradiol-inducible XVE:FBN5-B
construct was used to transform FBN5/fbn5-1 plants, and the
fbn5-1 lines were selected with PCR genotyping. These transgenic plants survived when germinated in soil because of the
leaky expression of XVE:FBN5-B, despite the absence of b-estradiol
(Supplemental Figure 7A). Two independent T4 homozygous
lines, XVE:FBN5-B#12 and XVE:FBN5-B#17, were characterized
and used for HPLC analysis. The XVE:FBN5-B#12 and #17
transgenic plants were clearly smaller, with slower growth rates,
than the wild-type plants (Supplemental Figures 7A to 7C). The
growth of the complemented T4 homozygous line (fbn5-1+35S:
FBN5-B) and a T4-overexpressing line of wild-type plants
(WT+35S:FBN5-B) was similar to that of the wild-type plants
(Supplemental Figures 7A to 7C). The rosette size of the plants is
determined during the course of plant development, and we
measured the fresh weight of the plants that grew to maturity
(33 d old). The fresh weights of the fbn5-1+35S:FBN5-B and
WT+35S:FBN5-B transgenic plants did not differ significantly
from those of the wild-type plants, whereas the fresh weights of
the XVE:FBN5-B#12 and #17 transgenic plants were reduced by
68 and 83%, respectively (Supplemental Figures 7B and 7C). The
contents of PQ-9, tocochromanols, carotenoids, and chlorophylls
were measured in the leaves of 6-week-old plants grown in soil
(Figure 3; Supplemental Figure 5). The levels of PQ-9 were reduced
;3- and 5-fold, and PC-8 was reduced nearly 9- and 38-fold in the
XVE:FBN5-B#12 and #17 transgenic plants, respectively (Figure
3C). The tocopherol contents of the XVE:FBN5-B#12 and #17
transgenic plants did not differ significantly from those of the wildtype plants (Figure 3D). However, the levels of PQ-9 and PC-8 in
the fbn5-1+35S:FBN5-B and WT+35S:FBN5-B transgenic plants
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Figure 4. Interaction Domain Mapping of FBN5 with SPS1 and SPS2.
(A) Schematic diagrams of the bait constructs of FBN5-B variants and full FBN5-A fused to the GAL4 DNA binding domain. The numbers represent
amino acid positions at the start and stop positions of the constructs. Full-length FBN5 contains chloroplast-targeting sequences and a-helix and
b-sheet structures.
(B) Specificity of the FBN5 interaction with either mature SPS1 or mature SPS2 in the Y2H system. Interactions of FBN5 variants with either mature
SPS1 or mature SPS2 fused to the GAL4 DNA-activation domain were examined in each yeast transformant (PBN204) on growth-selective
medium lacking Leu and Trp (SD-LW) or on selective medium also lacking Ura (SD-LWU) or Ade (SD-LWA). b-Galactosidase activity was tested in
each colony on SD-LW medium. Positive control was yeast transformed with the PTB (polypyrimidine tract binding protein) bait plasmid and PTB
prey plasmid (+/+). PTB is a homodimeric protein. Negative control was a cell transformed with the parental bait vector (pGBKT7) and prey vector
(pGADT7).
were similar to those in the wild-type plants (Figures 3C). Notably,
in the XVE:FBN5-B#12 and #17 transgenic plants, the reduced
contents of PQ-9 and PC-8 coincided with reduced levels of the
FBN5 transcript (Figure 3C; Supplemental Figure 7D). However,
the increased levels of FBN5 transcripts, by ;12-fold and 6-fold in
the fbn5-1+35S:FBN5-B and WT+35S:FBN5-B transgenic plants,
respectively, did not affect the accumulation of PQ-9 or PC-8
(Figure 3; Supplemental Figure 7D). The total carotenoid contents
did not differ between the plants. However, the XVE:FBN5-B#12
and #17 transgenic plants had more antheraxanthin compared
with the wild-type plants and fbn5-1+35S:FBN5-B and WT+35S:
FBN5-B transgenic plants, and so must have been photostressed
under these growth conditions (Supplemental Figure 5C). Taken
together, our results for the knockout and knockdown mutants of
FBN5 Is Essential for Synthesis of PQ-9
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Figure 5. Y2H Experiments to Test Interactions between FBN5-B and SPS1, SPS2, SPS3, VTE2, and HST Enzymes.
Either the FBN5-B preprotein (full) or mature FBN5-B (62-end) was fused to the GAL4 DNA binding domain (pGBKT7 vector). Either the preprotein (full) or
mature form of SPS1, SPS2, SPS3, VTE2, or HST was fused to GAL4 DNA-activation domain (pGADT7 vector).
(A) Transformed yeasts (PBN204) were dropped onto selective medium lacking Leu and Trp (SD-LW) or selective medium also lacking Ura (SD-LWU) or Ade
(SD-LWA). b-Galactosidase activity in each colony was tested on SD-LWA medium. Positive control was yeast transformed with the PTB bait plasmid and PTB
prey plasmid (+/+). PTB is a homodimeric protein. Negative control was a cell transformed with the parental bait vector (pGBKT7) and prey vector (pGADT7).
(B) The interaction strength test of FBN5-B with SPS1, SPS2, SPS3, VTE2 and HST. The transformants (AH109) were dropped onto selective medium also
lacking His (SD-LWH) but containing 5 or 15 mM 3-amino-1,2,3-triazole (3-AT), a competitive inhibitor of the yeast HIS3 protein.
FBN5 suggest that FBN5 is involved in the biosynthesis of PQ-9
and PC-8 in Arabidopsis leaves.
FBN5-B Interacts with SPSs Involved in PQ-9 Biosynthesis
FBN5 lacks obvious enzyme-associated protein domains, suggesting that it plays a structural role in PQ-9 biosynthesis. Accordingly, we tested whether FBN5 interacts with the proteins involved in
the PQ-9 biosynthetic pathway using the yeast two-hybrid (Y2H)
system. After confirming that the fusion of the full-length FBN5-B
cDNA to the GAL4 DNA binding domain (BD fusion) did not cause
self-transcription, this fusion protein was used as the bait to screen
Arabidopsis cDNA prey libraries. From a total of 1.24 3 107 yeast
transformants, 20 independent colonies were positive for lacZ activation and URA3 and ADE2 autotrophy. DNA sequence analysis
revealed that all 20 cDNA fusions encoded the mature portion of
SPS1 (At1g78510), lacking the first 60 N-terminal amino acid residues of the polypeptide. Arabidopsis has two paralogous solanesyl
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The Plant Cell
Figure 6. Interaction of FBN5-B with SPS1 and SPS2 in Chloroplasts.
(A) Colocalization of FBN5B with SPS1 or SPS2. Representative confocal
images are shown of the GFP and mRFP signals in FBN5B-mRFP Arabidopsis protoplasts transformed with either SPS1-GFP or SPS2-GFP.
(B) Immunoblot analysis of SPS1 and SPS2 using anti-HA antibody (upper
panel, input). Arabidopsis protoplasts transiently expressing FBN5B-GFP
with either SPS1-HA or SPS2-HA. Coimmunoprecipitated SPS1 and SPS2
were detected with an anti-HA antibody (lower panel, Co-IP).
(C) BiFC analysis of epidermal cells from N. benthamiana. FBN5B-VenusN
interacted with SPS1-SCFPC and SPS2-SCFPC (upper panels), but
FBN5A-VenusN did not interact with the fusion proteins (middle panels).
Negative and positive controls are shown in the lower left and right panels,
respectively. Chloroplasts were visualized with the autofluorescence of
chlorophyll. Bars = 20 µm.
diphosphate synthases, SPS1 and SPS2 (At1g17050), and these
proteins assemble the prenyl chain of PQ-9 in plastids (Jun et al.,
2004; Block et al., 2013). Because SPS1 and SPS2 share 80%
identical residues (Hirooka et al., 2005), the interaction between
the preprotein of FBN5-B (full-length peptide) and SPS2 was also
examined. Although mature SPS2 showed a strong interaction
with the preprotein of FBN5-B, the preprotein of SPS2 barely
interacted with the FBN5-B preprotein (Supplemental Figure 8). The
SPS1 preprotein also did not interact with the FBN5-B preprotein
(Supplemental Figure 8). These results indicate that the transit
peptides of SPS1 and SPS2 abolish their interactions with FBN5-B.
To further characterize the specificity and structural requirements for the interaction between FBN5-B and SPS, the interactions between different variants of FBN5-B and mature SPS1 or
mature SPS2 were analyzed. The construction of the FBN5-B
variants was based on the transit peptide, a-helix domain, and
b-sheet domain of the mature protein using a structural algorithm
(http://www.compbio.dundee.ac.uk/www-jpred/) (Figure 4A). The
results clearly indicated that the transit peptide, a-helix domain,
and b-sheet domain of FBN5-B alone did not interact with any
SPS, whereas the FBN5-B preprotein and mature FBN5-B did
interact with SPS. However, the FBN5-A preprotein, a product of
alternative splicing, did not interact with either mature SPS1 or
mature SPS2. These results suggest that the overall integrity and
the C-terminal region of FBN5-B are important for its interaction
with the SPS proteins (Figure 4B). The transit peptide of FBN5-B
did not abrogate the interaction with the SPS proteins. The failure
of overexpressed FBN5-A to interact with the SPS proteins in the
Y2H analysis or to complement the seedling-lethal fbn5-1 plants
supports the conclusion that the interaction between FBN5-B and
the SPS proteins is essential for plant growth.
Because it is possible that the enzymes involved in the biosynthesis of PQ-9 and the tocopherols form a multienzyme system
that functions in metabolic channeling, we examined the potential
interactions of FBN5-B with eight other proteins: SPS3, fanesyl
diphosphate synthase 1 (FPS1), FPS2, geranylgeranly diphosphate synthase 1 (GGPS1), VTE2, VTE3, HST, and geranylgeranly
reductase (GGR), which are metabolically connected (Supplemental
Figure 9A). However, none of them interacted with the FBN5-B
preprotein (Supplemental Figure 9B), which may have been because the preprotein form of each enzyme was used as the prey.
Therefore, we tested the binding of FBN5-B to mature SPS3,
VTE2, and HST because either their substrates or products are
highly hydrophobic (Figure 5). Both the Y2H analyses in yeast
strains PBN204 (Figure 5A) and AH109 (Figure 5B) clearly showed
that neither the FBN5-B preprotein nor mature FBN5-B interacted
with either the preprotein or mature forms of SPS3, VTE2, or HST.
A test of the intensity of the interaction between the proteins
in AH109 cells on medium containing 3-amino-1,2,3-triazole,
a competitive inhibitor of the yeast HIS3 protein, revealed that the
interactions between FBN5-B and the mature forms of SPS1 and
SPS2 were stronger than its interactions with the positive control
proteins (Figure 5B). It is noteworthy that FBN5-B did not interact
with SPS3, another SPS, responsible for the solanesyl moiety of
ubiquinone-9 in Arabidopsis (Ducluzeau et al., 2012). In summary,
both the preprotein and mature form of FBN5-B specifically interacts with mature SPS1 and mature SPS2, but not with other
enzymes involved in the biosynthesis of hydrophobic compounds,
FBN5 Is Essential for Synthesis of PQ-9
Figure 7. Transcript Levels of Genes Involved in Prenylquinone Biosynthesis in Wild-Type and fbn5-1 Plants.
(A) Comparative expression of 10 genes involved in prenylquinone biosynthesis, with ACT7 control. Expression was measured via microarray
analysis.
(B) Determination of relative expression of FBN5, SPS1, and SPS2 in wildtype and fbn5-1 plants with real-time RT-PCR. RNA samples were extracted from 3-week-old wild-type and fbn5-1 homozygous mutant
seedlings grown on MS medium + 1% sucrose. ACT7 expression was used
for normalization. Significant differences in gene expression in the wildtype and fbn5-1 plants were determined with Student’s t test. Data are
means 6 SE (n = 4).
suggesting that FBN5-B plays a role associated with SPP
biosynthesis.
FBN5-B Interacts with SPS in Chloroplasts
We demonstrated above that FBN5-B localizes to the chloroplasts (Figure 2). Our data for the transient expression of SPS1GFP and SPS2-GFP in protoplasts (Supplemental Figure 10),
together with previous studies (Jun et al., 2004; Block et al., 2013),
indicate that both SPS1 and SPS2 are targeted to the chloroplasts.
To further examine the colocalization of FBN5-B with SPS1 or SPS2,
their full-length cDNAs were cloned in frame with modified RFP
(mRFP) under the control of the CaMV 35S promoter. The resulting
construct, 35S:FBN5B-mRFP, also containing either 35S:SPS1-GFP
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or 35S:SPS2-GFP, was transiently coexpressed in Arabidopsis
protoplasts. SPS1-GFP colocalized with FBN5B-mRFP in the
chloroplasts, evident as overlapping green and red fluorescence
(Figure 6A). Although some green fluorescence associated with
SPS2-GFP appeared in the envelope membrane and cytosol, it was
primarily located in the chloroplasts and colocalized with FBN5BmRFP (Figure 6A). The signal for SPS2-GFP in the envelope membrane and in the cytosol could be attributable to its overexpression.
We next tested whether FBN5-B interacts with SPS1 or SPS2 in
planta using coimmunoprecipitation (Co-IP) and bimolecular
fluorescence complementation (BiFC) assays. In the Co-IP assay,
total protein extracts from Arabidopsis protoplasts coexpressing
GFP-tagged FBN5-B and either hemagglutinin (HA)-tagged SPS1
or HA-tagged SPS2 were incubated with anti-GFP affinity agarose. The immunoprecipitated proteins were detected with immunoblotting using an anti-HA antibody. As shown in Figure 6B,
FBN5-B coimmunoprecipitated with SPS1 and SPS2, which
suggests that these proteins interact in planta.
The BiFC analysis confirmed the in planta interactions between
FBN5-B and SPS1 or SPS2 in the chloroplasts (Figure 6C). In this
experiment, epidermal cells from Nicotiana benthamiana leaves
were transiently cotransformed with six different binary constructs
expressed under the CaMV 35S promoter. Two days after infiltration, green granular signals were detected in the chloroplasts
when FBN5-B-VenusN was paired with either SPS1-SCFPC or
SPS2-SCFPC, indicating their physical interaction in vivo (Figure
6C; Supplemental Movies 1 and 2). However, no green signal was
observed when FBN5-A-VenusN was paired with SPS1-SCFPC or
SPS2-SCFPC (Figure 6C), indicating that SPS1 and SPS2 interact
specifically with FBN5-B, but not with FBN5-A. Based on our
results, we conclude that FBN5-B interacts physically with SPS1
and SPS2 in chloroplasts. However, we note that to confirm these
findings, the proteins must be stably expressed in Arabidopsis
under their endogenous promoters.
Expression of Tocochromanols and PQ-9 Biosynthesis
Genes Is Not Altered in fbn5-1
To determine whether the PQ-9 deficiency in the fbn5-1 plants is
attributable to perturbation in the transcript abundance of the
genes encoding the enzymes for tocochromanols and PQ-9
biosynthesis, we monitored the expression levels of GGPS1, HST,
VTE2, VTE1, VTE3, VTE4 (g-tocopherol methyltransferase), SPS1,
and SPS2 with microarray data in wild-type and fbn5-1 plants. The
FPS1 and SPS3 genes, which are involved in the biosynthesis of
the solanesyl moiety of ubiquinone-9 in Arabidopsis, were also
included. As shown in Figure 7A, the transcript abundances of
these genes did not differ significantly in the wild-type and fbn5-1
plants. Using real-time PCR, we also confirmed that there were no
differences in the transcription levels of SPS1 and SPS2 in the
wild-type and fbn5-1 plants, except for the knocked-out FBN5
gene (Figure 7B). These results indicate that the deficiency of PQ-9
in fbn5-1 plants is specifically associated with FBN5.
FBN5-B-Deficient Plants Are Sensitive to Cold Stress
To determine the effects of FBN5 deficiency on photosynthesis,
various photosynthetic parameters were measured in wild-type
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in the XVE:FBN5-B transgenic plants under cold stress, but was
fully restored after their transfer to normal conditions (Figure 8A).
During cold stress, the defect in FBN5 primarily affected the
photochemical efficiency (ФPSII) and slightly reduced the lightdependent thermal dissipation component of nonphotochemical
quenching (ФNPQ) (Figure 8B). H2O2 was assayed by visualizing the
amount of precipitate formed after incubation with diaminobenzidine
(DAB) solution. There were no differences in DAB staining before
and after cold stress in the wild-type plants. However, cold stress
treatment significantly increased DAB staining in the XVE:FBN5B#17
leaves (Figure 8C).
DISCUSSION
Figure 8. Effects of Short-Term Cold Stress on Photosynthetic Properties
of PSII and Hydrogen Peroxide Production in Wild-Type and XVE:FBN5B
Leaves.
Plants grown for 4 weeks at 22°C, with a 16/8-h light/dark photoperiod at
100 mmol m–2 s–1 illumination were considered to be cultivated under
normal conditions. Plants were transferred to 5 to 7°C with a 16/8-h light/
dark photoperiod under 60 mmol m–2 s–1 illumination for 5 d and then
transferred to normal conditions to recover.
(A) Fv/Fm measurements after each treatment. Data are means 6 SD (n = 3).
(B) Fraction of absorbed light consumed by photochemistry (ФPSII), ΔpHand xanthophyll-regulated thermal dissipation (ФNPQ), and the sum of
fluorescence and constitutive thermal dissipation (ФF,D). Asterisks denote
significant differences between wild-type and XVE:FBN5B transgenic plants
by Student’s t test: *P < 0.05 and ***P < 0.001. Data are means 6 SD (n = 3).
(C) H2O2 detected in wild-type and XVE:FBN5B#17 leaves from normal and
cold-stressed plants, determined with DAB solution staining.
and XVE:FBN5-B transgenic plants before their exposure to cold
stress, after a 5-d period of cold stress, and after their transfer to
normal temperatures. The maximum quantum efficiency of PSII
photochemistry (Fv/Fm) was unchanged in the wild-type plants
subjected to cold stress. However, Fv/Fm decreased significantly
We characterized an fbn5-1 T-DNA insertion mutant to determine
how a defect in FBN5, a structural lipid-associated protein, produces a seedling-lethal phenotype. Our study is not the first to
report a seedling-lethal phenotype for the fbn5-1 T-DNA insertion
mutant. Savage et al. (2013) found that FBN5 is an essential
nuclear gene encoding a protein that targets the chloroplast, although they did not study the gene in depth. Consistent with our
results (Figure 1), segregating seeds from parent plants heterozygous for fbn5-1 yielded no homozygotes that could survive in
soil. Furthermore, the seedlings homozygous for the fbn5-1 allele
were small and pale green, with or without sucrose supplementation (Savage et al., 2013). In our study, we confirmed that the
FBN5 mutation is responsible for the lethal phenotype by
complementing fbn5-1 plants with 35S:FBN5-B (Supplemental
Figure 3).
The fbn5-1 plants and previous findings with the pds2 mutant
(Norris et al., 1995; Tian et al., 2007) and sps1 sps2 double mutant
(Block et al., 2013) demonstrated the absolute requirement for
PQ-9 during plant development. Phytoene desaturase requires
quinones and molecular oxygen as electron acceptors to pass
electrons from phytoene (Mayer et al., 1990). An Arabidopsis pds2
(HST) mutant accumulated phytoene but not downstream carotenoids, indicating that PQ-9 is an essential component of phytoene desaturation (Norris et al., 1995). However, in our study,
fbn5-1 and pds2-1 (a leaky mutant of pds2) plants biosynthesized
downstream carotenoids without accumulating phytoene, although the total carotenoid levels were reduced relative to that in
wild-type plants (Supplemental Figure 5A and Supplemental Table
2). It is likely that even the extremely low levels of PQ-9 in the fbn5-1
and pds2-1 plants were sufficient for phytoene desaturation,
but resulted in reduced carotenoid biosynthesis (Figure 3A;
Supplemental Tables 1 and 2). The similar carotenoid levels in
the XVE:FBN5-B transgenic and wild-type plants implies that
a certain level of PQ-9 is sufficient for phytoene desaturase
activity (Supplemental Figure 5C). The lower photosynthetic
efficiency and higher levels of H2O2 observed in the fbn5-1 plants
(Supplemental Figure 4) and the enhanced sensitivity to cold in
the XVE:FBN5-B transgenic plants (Figure 8) are probably attributable to the deficiency in PQ-9 biosynthesis. A limited PQ
pool reduces the forward steps in electron transport, resulting in
a reduced state of QA. This generates reactive oxygen species
and highly oxidized radicals, which damage PSII (Napiwotzki
et al., 1997; Vass, 2012). Under cold stress, the PSII excitation
pressure is higher than under normal growth conditions (Wise,
FBN5 Is Essential for Synthesis of PQ-9
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Figure 9. Proposed Model for the Biosynthesis of PQ-9 and PC-8 in Chloroplasts.
SPS of the FBN5-B/homodimeric SPS complex binds to GGPP and IPP and then condenses five molecules of IPP to GGPP, yielding SPP. Synthesized SPP
is removed from SPS when it binds to FBN5-B. SPP released from FBN5-B or SPP bound to FBN5-B is condensed with HGA by HST, generating MSBQ.
MSBQ is converted to PQH2-9 by VTE3. VTE1 cyclizes PQH2-9 to PC-8 in plastoglobules.
1995; Gray et al., 2003; Ensminger et al., 2006). Therefore, the
lower levels of PQ-9 in the XVE:FBN5-B transgenic plants might
increase the proportion of “closed” PSII reaction centers under
cold stress relative to that under normal growth conditions,
resulting in lower Fv/Fm and the greater accumulation of H2O2 in
the leaves.
As shown in Figure 3, the amounts of PQ-9 and PC-8 were
significantly reduced in both the FBN5 knockout and knockdown
plants, whereas the levels of total tocopherols were not affected
(Figure 3). PQ-9 and tocopherols both have homogentisate head
groups. These results indicate that the homogentisate synthesis in
the fbn5-1 plants was normal. Therefore, we conclude that FBN5 is
specifically involved in the biosynthesis of the solanesyl moiety
and/or the transfer of the solanesyl moiety to homogentisate.
Block et al. (2013) demonstrated that SPS1 and SPS2 are functionally redundant in PQ-9 biosynthesis. Consistent with that
finding, we have shown that FBN-5B interacts with both SPS1
and SPS2 (Figures 4 and 6), thus explaining the similar phenotypes
of the fbn5-1 plants and the sps1 sps2 double mutant plants
(Figures 1 and 3). It is unlikely that FBN5-B forms a complex with
HST that functions in PQ-9 biosynthesis because FBN5-B did not
interact with HST (Figure 5). However, we cannot exclude the
possibility that biosynthesis of PQ-9 occurs by metabolic channeling because the interaction between SPS1 or SPS2 and HST
was not examined. It must be noted that FBN5-B did not interact
with other enzymes involved in the biosynthesis of hydrophobic
compounds, such as tocochromanols or benzoprenylquinones
(Figure 5; Supplemental Figure 9). Furthermore, the fbn5-1 mutation
did not alter the transcript levels of the genes involved in the synthesis
of tocochromanols or benzoprenylquinones. Specifically, the
expression of SPS1, SPS2, and HST, which are directly responsible for PQ-9 biosynthesis, did not fluctuate in the fbn5-1
plants (Figure 7). These results confirm that the binding of FBN5-B
to SPS1 and SPS2 is essential for PQ-9 biosynthesis.
What role does the physical binding of FBN5-B to SPS play in
PQ-9 biosynthesis? It has been reported that the long-chain
polyprenyl pyrophosphate synthase often requires detergent or
another factor for its optimal activity because its product release
is slow. Several in vitro studies have demonstrated that longchain trans-polyprenyl diphosphate synthases from several organisms require the addition of phospholipids or detergent to the
assay buffer to enhance their product release and maintain efficient turnover (Pan et al., 2013). The activity of SPS isolated from
Micrococcus luteus was enhanced in the presence of BSA or
a high molecular mass fraction (HMF) separated from cell-free
extracts of M. luteus (Ohnuma et al., 1991). This suggests that
HMF contains a factor that binds to the polyprenyl products and
removes them from the active site of the enzyme to facilitate
and maintain the turnover of catalysis. The steady state rate of
octaprenyl pyrophosphate synthase purified from Escherichia coli
also accelerated 3-fold in the presence of Triton X and HMF (Pan
et al., 2002). Recently, mitochondrial coenzyme Q9 was reported
to be a lipid binding protein that associates physically with coenzyme Q7 (Lohman et al., 2014). The authors suggested that
COQ9 binds immature COQ species and presents them to COQ7
to allow coenzyme biosynthesis. Singh and McNellis (2011) showed
that lipocalin motif 1 is conserved among the Arabidopsis fibrillins
and suggested that the fibrillin family is involved in binding to and
transporting small, hydrophobic molecules. Consistent with the
lipocalin activity of the fibrillins, it has been suggested that FBN4 is
involved in trafficking PQ-9 and other small hydrophobic molecules from the thylakoids to the plastoglobules (Singh et al., 2012).
FBN5 contains a possible lipocalin motif 1, which encompasses
amino acid residues 131 to 142 (Supplemental Figure 1) (Singh and
McNellis, 2011). Based on these studies, we conclude that FBN5B is involved in the biosynthesis of the solanesyl moiety, insofar
as the lipid binding domain of FBN5-B binds to the hydrophobic
solanesyl moieties that are generated by SPS1 and SPS2. Thus,
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The Plant Cell
FBN5-B stimulates the enzymatic activity of SPS1 and SPS2.
When FBN5-B is deficient, the release of the solanesyl moiety from
the enzyme and the enzyme turnover become slower, delaying
PQ-9 biosynthesis. However, to support our hypothesis, in vitro
experiments are required to determine whether the presence of
FBN5-B enhances the reaction efficiency of SPS1 or SPS2.
Our work has shown that a plastid-lipid-associated protein,
FBN5, is required for PQ-9 biosynthesis through its physical and
functional interactions with SPS1 and SPS2 in Arabidopsis. The
PQ-9 biosynthetic enzymes are well characterized, and our work
presents FBN5 as a new component of the PQ-9 biosynthetic
process. We have also shown that at least one member of the
fibrillin family is physically and functionally associated with other
enzymes. Therefore, given the plastid-lipid binding domain in the
fibrillins, future studies that clarify the relationships between
fibrillins and other enzymatic reactions, especially those producing hydrophobic molecules, will be exciting. We propose
a model for PQ-9 biosynthesis in Arabidopsis based on our results
and data in the literature (Figure 9). In our model, SPS of the FBN5B/dimeric SPS complex catalyzes the sequential condensation
of five molecules of IPP with GGPP to yield the all-trans form of
SPP. FBN5-B of the FBN5-B/dimeric SPS complex then removes SPP from the catalytic site of SPS. The SPP released from
FBN5-B or the SPP bound to FBN5-B is condensed with HGA by
HST to produce MSBQ. Finally, MSBQ is converted to PQH2-9 by
VTE3, followed by its translocation to the thylakoids and plastoglobules, and some PQH2-9 is cyclized to PC-8 by VTE1 in the
plastoglobules.
the Gateway transformation vector pENTR/D-TOPO (Invitrogen) with the
TOPO reaction, according to the manufacturer’s instructions. pENTRFBN5-A and pENTR-FBN5-B were recombined with LR reactions into the
plant transformation vector, pB2GW7.0, where FBN5-A and FBN5-B were
controlled by the CaMV 35S promoter (Karimi et al., 2002). Competent
Agrobacterium tumefaciens cells (GV3101) were transformed with the
constructs pB2GW7.0-FBN5-A and pB2GW7.0-FBN5-B with freeze-thaw
methods. Heterozygous FBN5/fbn5-1 Arabidopsis plants were transformed with the Agrobacterium described above, using the floral dip
transformation method (Clough and Bent, 1998), and selected by their
resistance to BASTA (Bayer). Genomic DNA was extracted from the leaves
of the BASTA-resistant plants and used to identify the homozygous fbn5-1
plants. pENTR-FBN5-B was recombined with the LR reaction into the
pMDC7 destination vector (Zuo et al., 2000) to be expressed under the
control of the XVE b-estradiol-inducible promoter and was then used to
transform heterozygous FBN5/fbn5-1 Arabidopsis plants, which were
screened as described above.
Histochemical Analysis of GUS Expression
A 2.7-kb region of the FBN5 genomic DNA including the 59-untranslated
region was amplified and cloned into pENTR/D-TOPO and then transferred
into the pMDC163 destination vector with the LR reaction (Curtis and
Grossniklaus, 2003). Arabidopsis was transformed with the construct, as
described above, and the transgenic plants were selected by their hygromycin resistance. The plant tissues were vacuum infiltrated with GUS
staining buffer, as described previously (Kim et al., 2008). The GUS staining
buffer contained 50 mM sodium phosphate buffer (pH 7.0), 0.1% Triton
X-100, 2 mM potassium ferrocyanide, 10 mM EDTA, 100 µg mL–1 chloramphenicol, and 1 mg mL–1 5-bromo-4-chloro-3-indolyl-b-D-glucuronic
acid. The infiltrated tissues were incubated at 37°C overnight. The nonspecific stains and colored pigments were then removed with 70% ethanol.
METHODS
Microarray and qRT-PCR Analyses
Plant Materials and Growth Conditions
Mutants fbn5-1 (SALK_064597), pds2-1 (SALK_024357), aae14-1 (SALK_
060226), gdc1-2 (SALK_151530C), and sbp-1 (SALK_090549C) were
acquired from the ABRC. Arabidopsis thaliana ecotype Columbia-0 was
grown in soil or on agar plates containing 0.53 MS medium supplemented with/without sucrose under a 16-h-light/8-h-dark photoperiod
with 100 µmol m–2 s–1 fluorescent light at 22°C. Nicotiana benthamiana
(tobacco) plants were grown under a 16-h-light/8-h-dark photoperiod at
23°C under 100 µmol m–2 s–1 fluorescent light. Arabidopsis leaf tissues
were genotyped with PCR using genomic DNA as the template. The
primers used in this study are listed in Supplemental Table 3.
Affymetrix GeneAtlas Arabidopsis Gene 1.1 ST Array Strips were used to
comparatively analyze the genes expressed in the wild-type and fbn5-1
seedlings. Four biological replicates of total RNA were prepared from
3-week-old wild-type and fbn5-1 seedlings. Probe preparation, hybridization, and the signal normalization process were as described by Lee et al.
(2008). The raw microarray data were deposited in the Gene Expression
Omnibus under accession number GSE68275 (http://www.ncbi.nlm.nih.
gov/geo).
Real-time PCR was performed with SYBR Green Premix Ex Taq II
(Takara) and the CFX96 Touch Real-Time PCR detection system (Bio-Rad
Laboratories), as specified by the manufacturer. The primers for real-time
qRT-PCR are listed in Supplemental Table 3.
RT-PCR Analysis
Total RNA was extracted from the leaf tissues using TRIzol reagent (SigmaAldrich) according to the manufacturer’s instructions. cDNA was synthesized from the total RNA using the PrimeScript 1st Strand cDNA
synthesis kit (Takara), as recommended by the manufacturer. The PCR
reactions were performed with Takara Ex Taq DNA Polymerase and the
appropriate primer pairs. The PCR cycling parameters were (1) denaturation, 30 s at 94°C; (2) annealing, 30 s at 58°C; and (3) extension, 60 s
at 72°C. The reactions were run for 32 cycles.
Complementation of the Arabidopsis fbn5-1 Mutation
The full-length FBN5 coding sequences (FBN5-A and FBN5-B) were
amplified from cDNA obtained from Arabidopsis leaves using KOD Hot
Start DNA Polymerase (Novagen). The amplified products were cloned into
Y2H Screen and Interaction Assay
The FBN5-B coding region was amplified by PCR with primers containing
restriction sites and was cloned in frame between the EcoRI and BamHI
sites of the bait vector (pGBKT7) to form pGBKT7-FBN5-B. Y2H screening
of FBN5-B was performed in yeast strain PBN204 containing three reporter
genes (URA3, lacZ, and ADE2) under the control of different GAL promoters. Yeast transformants containing the FBN5-B bait and an Arabidopsis cDNA AD library were plated on selection medium (SD-leucine,
tryptophan, uracil [SD-LWU]) that supported the growth of yeast cells
containing both bait and prey plasmids, thus yielding proteins that interacted with each other. After the yeast colonies were selected on uracildeficient medium, we monitored the activity of b-galactosidase. The URA+
and lacZ+ colonies were also examined on adenosine-deficient medium.
To construct the Y2H systems, the full-length coding sequences of the
FBN5 Is Essential for Synthesis of PQ-9
following Arabidopsis genes were amplified from the cDNA from Arabidopsis leaves: SPS1 (At1g78510), SPS2 (At1g17050), SPS3 (At2g34630),
FPS1 (At5g47770), FPS2 (At4g17190), GGPS1 (At4g36810), VTE2
(At2g18950), VTE3 (At3g63410), HST (At3g11945), and GGR (At4g38450).
The coding sequences for SPS1, SPS2, SPS3, VTE2, and HST, without the
chloroplast transit peptide sequences, were also amplified. The amplified
products were cloned into the pGADT7 vector (prey). The bait (pGBKT7FBN5-B) and prey plasmids were introduced into two yeast strains:
PBN204 (containing URA3, ADE2, and lacZ as reporters) and AH109
(containing HIS3, ADE2, and lacZ). The transformants were spotted onto
SD-LW medium. After incubation for 3 d at 30°C, the colonies were
replica-plated onto several selective media. To test the interactions between
the FBN5 variants and either mature SPS1 or mature SPS2, the corresponding sequences of the FBN5 variants were cloned into pGBKT7. The
coding sequences for SPS1 and SPS2, without their transit peptide sequences, were cloned into pGADT7. The Y2H experiment was performed as
described above.
Subcellular Localization
The FBN5-A, FBN5-B, SPS1, and SPS2 full-length coding regions, each
containing a BamHI site, were amplified from the cDNA of Arabidopsis
leaves using KOD Hot Start DNA Polymerase (Novagen). The amplified
FBN5-A sequence was fused in frame to the superfolder GFP (sGFP) gene
in vector 326sGFP and FBN5-B sequence was to the mRFP gene in the
vector 326mRFP and to the sGFP gene in vector 326sGFP. The amplified
SPS1 and SPS2 sequences were fused in frame to the sGFP gene in vector
326sGFP. For the transient expression of the proteins, mesophyll protoplasts were prepared from 3-week-old Arabidopsis plants and transformed
with the above-mentioned plasmids, as described previously (Jin et al.,
2001). About 1.5 3 106 protoplasts were transfected with 20 µg plasmid
DNA, and the transfected protoplasts were then incubated in the dark.
Fluorescence images of the protoplasts were taken with a fluorescence
microscope (Axioplan 2; Carl Zeiss) equipped with a 403/0.75 objective
(Plan-Neofluar) and a cooled CCD camera (Senicam; PCO Imaging). The
filter sets used were XF116 (exciter, 474AF20; dichroic, 500DRLP; emitter,
510AF23; Omega) for sGFP and XF33/E (exciter, 535DF35; dichroic,
570DRLP; emitter, 605DF50; Omega) for mRFP. The protoplasts were
harvested 24 h after transformation and resuspended in sonication buffer
(10 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 1 mM EDTA) and complete
protease inhibitor cocktail (Roche). The protoplasts were lysed with brief
sonication and centrifuged at 10,000g for 10 min to remove the debris.
The proteins were separated with SDS-PAGE and analyzed with immunoblotting with a monoclonal anti-GFP antibody (Clontech). The
protein blots were developed with protein gel blot detection solution
(Supex; Neuronex) and visualized with the LAS3000 image capture
system (Fuji Film).
BiFC
The entire coding regions of CNX6, FBN5-A, FBN5-B, SPS1, and SPS2
were cloned into the destination vectors pDEST-GWVYNE (Venus yellow
fluorescent protein amino acids 1 to 173) (CNX6, FBN5-A, and FBN5-B)
and pDEST-GWSCYCE (SCFP3A super cyan fluorescent protein amino
acids 156 to 239) (CNX6, SPS1, and SPS2). FBN5-B-VenusN and CNX6VenusC were used as a negative control; CNX6 proteins form a dimer in the
heterotetramer molybdopterin synthase complex (Gehl et al., 2009). CNX6VenusN and CNX6-VenusC were used as a positive control; a CNX6
homodimer is formed by the reconstitution of green signals from CNX6VenusN and CNX6-VenusC. We tested the following combinations for
interaction: FBN5-A-VenusN and SPS1-SCFPC, FBN5-A-VenusN and
SPS2-SCFPC, FBN5-B-VenusN and SPS1-SCFPC, and FBN5-B-VenusN
and SPS2-SCFPC (Gehl et al., 2009). The constructs were transiently introduced into the leaves of N. benthamiana by Agrobacterium GV3103
13 of 16
infiltration (Gehl et al., 2009). The plants were maintained in the dark for
2 d and then analyzed with confocal microscopy under CLSM (Nikon C2
confocal microscope).
Co-IP
The FBN5-B full-length coding region, containing XbaI and BamHI sites,
was fused in frame to the HA gene cloned in vector 326HA vector (Min et al.,
2007). FBN5-B-HA was transiently expressed with either SPS1-sGFP or
SPS2-sGFP in mesophyll protoplasts of Arabidopsis, and proteins were
extracted from protoplasts as described above. Protein extracts in immunoprecipitation buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM
EDTA, 1 mM EGTA, 0.5% Triton X-100, and complete protease inhibitor
cocktail [Roche]) were incubated with an anti-GFP antibody (Clontech) for 3
h at 4°C and then incubated with protein A-Sepharose beads (Amersham)
for 1 h at 4°C. The beads were washed three times with immunoprecipitation buffer. After washing, the proteins were released by incubation for 10
min in SDS sample buffer at 98°C and analyzed with immunoblotting using
an anti-HA (Roche) or anti-GFP antibody (Clontech).
Measurement of Photosynthetic Parameters
The maximal photochemical activity of PSII (Fv/Fm) was measured with
a portable chlorophyll fluorimeter (Walz) under atmospheric conditions.
Fv/Fm was measured in leaves that were dark adapted for 10 min and was
calculated as (Fm – Fo)/Fm, where Fo is the initial chlorophyll fluorescence
level and Fm is the maximal fluorescence level, determined with an intense
pulse of white light. The stress-dependent reduction in Fv/Fm values was
interpreted as the photoinhibition of PSII. The utilization of absorbed light
energy was measured with a pulse amplitude modulation fluorimeter (PAM
101; Walz) on 4-week-old plants, as described previously (Hendrickson
et al., 2004). After dark adaptation for 10 min, Fo and Fm were measured, and
then the steady state chlorophyll fluorescence, Fs, was determined under
illumination with 700 µmol m–2 s–1 actinic light.
Reactive Oxygen Species Determination
Three individual plants of each genotype were submerged in 20 mL DAB
(Sigma-Aldrich) staining solution (0.1% [w/v] DAB, pH 3.0, and 10 mM
NaH2PO4), introduced by vacuum infiltration. The DAB-infiltrated tissues
were then incubated in the dark at ambient temperature for 24 h (Daudi and
O’Brien, 2012).
HPLC Analysis
For the analysis of carotenoids, frozen tissues were ground in liquid nitrogen, and the pigments were extracted and analyzed with HPLC using
a Shimadzu LC-20AD chromatograph, as previously described (Tian and
DellaPenna, 2001). The HPLC peak areas at 440 nm were integrated.
Chromatography was conducted at 30°C on a C18 reverse-phase column
(5 µM Supelco Discovery C18 column, 150 3 4.6 mm). The chromatographic conditions were a flow rate of 1.0 mL/min with solvent A (acetonitrile:water = 9:1 v/v, plus 0.1% triethylamine) and solvent B (ethyl
acetate), using the following gradient: 0 to 5 min, 0 to 33.3% B; 5 to 13 min,
33.3 to 66.7% B; 13 to 13.2 min, 100% B; 13.2 to 15.9 min, 0% B. To analyze
PQ-9 and the tocochromanols, the total lipids were extracted from frozen
tissues, as described previously (Bligh and Dyer, 1959). The samples in
ethanol were analyzed by HPLC on a C18 column at 30°C and separated in
isocratic mode with methanol at a flow rate of 1.0 mL/min. PQ-9 was
detected spectrophotometrically at 255 nm. The tocopherols and PC-8
were detected fluorimetrically (290 nm excitation; 330 nm emission). The
compounds were quantified with their corresponding external calibration
standards, and the data were corrected by comparison with the recovery of
rac-Tocol (Matreya) as the internal standard.
14 of 16
The Plant Cell
Accession Numbers
Sequence data from this study can be found in the GenBank/EMBL libraries
under the following accession numbers: AAE14 (At1g30520), ACT2
(At3g18780), ACT7 (At5g09810), CNX6 (At2g43760), FBN1 (At4g04020/
At22240), FBN2 (At2g35490), FBN4 (At3g23400), FBN5 (At5g09820),
FPS1 (At5g47770), FPS2 (At4g17190), GDC1 (At1g50900), GGPS1
(At4g36810), GGR (At4g38450), HST/PDS2 (At3g11945), OsSPS2
(AK066579), PTB (CAA43973), SBP (At3g55800), SiSPS (DQ889204),
SPS1 (At1g78510), SPS2 (At1g17050), SPS3 (At2g34630), VTE1
(At4g32770), VTE2 (At2g18950), VTE3 (At3g63410), and VTE4
(At1g64970).
Supplemental Data
Supplemental Figure 1. Gene Structure of FBN5.
Supplemental Figure 2. Expression Patterns of FBN5.
Supplemental Figure 3. Genetic Complementation of the SeedlingLethal Phenotype of fbn5-1 Plants with FBN5-B cDNA.
Supplemental Figure 4. H2O2 Detection in Wild-Type and fbn5-1
Plants by Staining with DAB.
Supplemental Figure 5. Quantification of Carotenoids and Chlorophylls in Arabidopsis Leaves with Reverse-Phase HPLC.
Supplemental Figure 6. Phenotypes of Pale-Green Mutants and WildType Plants on 1% Sucrose-Supplemented MS Medium.
Supplemental Figure 7. Growth and Fresh Weight of FBN5 Transgenic Plants.
Supplemental Figure 8. Interactions of Preprotein and Mature SPS
with Full-Length FBN5-B.
Supplemental Figure 9. Enzymes Tested for Their Interaction with
FBN5-B Using Yeast Two-Hybrid Analysis.
Supplemental Figure 10. Subcellular Localization of SPS1 and SP2.
Supplemental Table 1. Quantification of Tocochromanols and Prenylquinones in the Leaves of Each Pale-Green Mutant.
Supplemental Table 2. Quantification of Carotenoids and Chlorophylls in the Leaves of Each Pale-Green Mutant.
Supplemental Table 3. Oligonucleotides Used in This Study.
Supplemental Movie 1. Interaction of FBN5-B and SPS1 in
Chloroplasts.
Supplemental Movie 2. Interaction of FBN5-B and SPS2 in
Chloroplasts.
ACKNOWLEDGMENTS
This work was supported by the National Academy of Agricultural Science
(Project PJ01007505), the Cooperative Research Program for Agricultural
Science and Technology Development (SSAC Grant PJ01108101), and the
Rural Development Administration, Republic of Korea.
AUTHOR CONTRIBUTIONS
E.-H.K. and H.U.K. designed the study, performed most of the experiments, and wrote the article. Y.L. performed the imaging analysis with
fluorescence microscopy, the SDS-PAGE and immunoblotting for protein
localization, and the Co-IP experiments. All authors discussed the results
and commented on the article.
Received August 7, 2015; revised September 17, 2015; accepted September 17, 2015; published October 2, 2015.
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Fibrillin 5 Is Essential for Plastoquinone-9 Biosynthesis by Binding to Solanesyl Diphosphate
Synthases in Arabidopsis
Eun-Ha Kim, Yongjik Lee and Hyun Uk Kim
Plant Cell; originally published online October 2, 2015;
DOI 10.1105/tpc.15.00707
This information is current as of June 17, 2017
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